Scanning optical microscope

ABSTRACT

An optical microscope suitable for scanning below the surface of specimens of low optical contrast and particularly for scanning buried tissues and cells. Optical means focus a beam of parallel light within the object and means are provided to scan by moving an objective lens system along to axes orthogonal to the optical axis. An image is generated in a cathode-ray tube.

llnited States Patent Davidovits et al.

SCANNING OPTICAL MICROSCOPE Inventors: Paul Davidovits, 95 LakeviewTerrace;

Maurice David Egger, 70 Edgewood Way, both of New Haven, Conn. 06515Filed: June 19, 1970 Appl. No.: 47,717

US. Cl ..l78/6.8, l78/DIG. 1, 250/495 A,

Int. Cl ..H04n 7/18, G02b 21/06 Field of Search 178/68, 7.6, 7.1, 7.2,DIG. l,

l78/DIG. 27, DIG. 35, 6; 250/495 A; 350/86, 91 I References Cited UNITEDSTATES PATENTS 3,460,880 8/1969 Henderson ..350/91 X 3,463,882 8/1969Herbold ..l78/7.lX

OTHER PUBLICATIONS Flying Spot Microscope, Young et al. ElectronicsJuly, 1953 pp. 137-139 Primary Examiner-Robert L. RichardsonAttorney--Bessie A. Lepper [57] ABSTRACT 19 Claims, 5 Drawing Figures ik i x 2 22 1 POWER SUPPLY l 45 I oscu ATOR AMPLI- W4 :fi I FIER 6 g lATOR I I8 AMPLI- FIER //47 i PHASE PHASE I 1 i SHIFTER 52 SHIFTER 53LASER SUPPLY POWER PAIENIEDFEB15 m2 SHEET 3 [If 3 &

OSCILLISCOPE Poul Dovidovits Maurice D. Egger INVENTO/PS Br Z W AHorneySCANNING OPTICAL MICROSCOPE This invention relates to an opticalscanning microscope and more particularly to one which is suitable formicroscopic observation of thick specimens of low optical contrast.

In many cases it would be highly desirable to be able to study in vivo athin layer of buried tissue or cells at conventional opticalmagnification. With present microscope techniques it is not possible,for instance, to make satisfactory observations of buried brain cells inliving animals. The study of these cells is, however, of fundamentalimportance to the understanding of many neurophysiological problems. Forexample, the ability to observe buried nerve cells should make itpossible to determine whether or not cells of this nature are altered bychanges in sensory and motor activity. It should also be possible forexample to determine whether cells of the visual cortex show any changein structure when bombarded by afferent impulses; to determine whetherthe cortical cells from which electrical recordings are being made showany changes in morphology correlated with various phases of theirelectrical activity; or whether it is possible to observe the actualsprouting of cut fibers or regeneration of cells in lower vertebrates.These are but a sampling of the type of information which is sought byanatomists, and which can possibly be answered by the optical scanningmicroscope of this invention.

The scanning optical microscope of this invention also offers a researchtool for observation of buried tissues other than cells; and may alsohave clinical applications other than observation and recordation ofanatomical phenomena. As an example, the apparatus of this invention mayuse ultraviolet light to treat tumors selectively and thus avoid muchdestructive surgery. It may also be used to diagnose and locate certaintypes of tumors of the central nervous system and to make diagnosticobservations of subcutaneous tissue and organs in intact organismswithout exploratory surgery.

The optical scanning microscope of this invention may also be applied toindustrial inspection such as the case of observing physicalinconsistencies or nonuniformities below the surfaces of such materialsas plastics and the like. The application of the optical scanningmicroscope of this invention is, of course, limited in this respect bythe character of the electromagnetic radiation which can be used sinceit must be capable of being focused and of penetrating through thematerial to be examined.

Conventional transmitted light microscopy, including phase andinterference microscopy, is unsuitable for observation of cells andtissue in vivo because of the thickness of the material to be examined.Moreover, enormous technical problems stand in the way of using electronmicroscopy for observation of living brain cells in vivo. Thus, somemodification of reflected light microscopy provides the best possibleapproach to the solution of such a problem. However, because lightreflected back into the microscope from many different layers of tissueor cells degrades the quality of the image of the object, conventionalreflected light microscopy is usually unsatisfactory with low contrasttranslucent material such as unstained brain tissue and the like.

An early attempt at solving this problem was the design and constructionof a scanning microscope in which the optical field was scanned by aperforated rotating disc (see for example Petran, l-ladravsky, Egger,and Galambos, A Tandem- Scanning Reflected Light Microscope," J. Opt.Soc. Amer., 58:661-664 (1968)). Although the instrument described inthis reference eliminated unwanted reflections and allowed theobservation of brain cells or similar tissue, the use of light wasinefficient in that no more than about 10 to 10" or less of the lightincident from the source traveled through the eyepiece to form an image.U.S. Pat. No. 3,013,467 describes an optical scanning microscope using asource of nonparallel light and achieving scanning by moving the objectto be examined. This arrangement requires that the object be rigidlymounted on a moving scanning platform, a highly impractical arrangementfor the examination of objects of any size.

Moreover, the point light source used in U.S. Pat. No. 3,013,467 limitsthe light energies obtainable in the device and hence the resolution andthe final scan obtainable. Other devices designed to obtainsubstantially the same objects in different ways as the microscope ofthis invention are described by Katsuki, Suga, Nomato and Nakatsubo inProc. Japan Acad., 37:588( l96l Egger and Petran in Science 157305-1307(1967); and by Katsuki and Kanno in Jap. J. Physiol. l8z391-402, (1968).Although, some of the devices described in this prior art have met withsome limited success, they have not developed into practical opticalscanning microscopes.

It is, therefore, a primary object of this invention to provide animproved optical scanning microscopeparticularly suitable forobservation of thick specimens of low optical contrast. It is anotherobject of this invention to provide an apparatus of the characterdescribed for in vivo observations of living cells, tissues, fibers,etc. Another object of this invention is to provide such an apparatuswhich permits the object to be examined to remain fixed, therebyincreasing the practical accuracy of the scan and eliminating thenecessity of mounting the object rigidly to a moving scanning platform.An additional object of this invention is to provide an apparatus whichcan use a wide range of the electromagnetic spectrum, i.e., from theultraviolet into the infrared. Yet another object is to provide such anapparatus which can use a laser as a source of parallel light thusmaking it possible to increase the range of light energy obtainable.Still another object is to provide an apparatus of the characterdescribed which is relatively simple to construct and operate. Otherobjects of the invention will in part be obvious and will in part beapparent hereinafter.

The invention accordingly comprises the features of construction,combinations of elements, and arrangement of parts which will beexemplified in the constructions hereinafter set forth, and the scope ofthe invention will be indicated in the claim.

For a fuller understanding of the nature and objects of the invention,reference should be had to the following detailed description taken inconnection with the accompanying drawings in which FIG. 1 is adiagrammatic view of the apparatus of this invention in a preferredembodiment;

FIG. 2 is a optical diagram showing the manner in which the focal pointof the lens system shifts when two objective lens are used and are movedto obtain the desired scanning pattern;

FIGS. 3 and 4 are diagrammatic views of a modification of the apparatusin FIG. 1 using only one objective lens in focusing the light into thesubject to be observed; and

FIG. 5 is a diagrammatic view of the scanning microscope of thisinvention adapted for transmission microscopy.

In the apparatus of this invention parallel radiant energy from asuitable source, e.g., a laser, is optically focused by a lens systemwithin the object to be scanned. The lens system is preferably movedalong two, mutually perpendicular axes orthogonal to the incidentparallel beam, within the scanning limits from about one-hundredth toone millimeter. The scanning directions will henceforth be called thex-axis and y-axis. It is also within the scope of this invention to movethe elements of the lens system orthogonal to the incident beam but notnecessarily in mutually perpendicular directions. The scanning motion issmall enough so that the total amount of incident parallel radiantenergy intercepted by the lens system is not appreciably altered in thecourse of scanning. The motion of the lens system in the x and ydirections causes the focused light to scan the desired region in theobject.

A fraction of the radiant energy reflected from the object isintercepted by the scanning lens system. Only the radiant energyreflected from the focal point is projected back into a parallel beam.Radiant energy reflected from other parts of the object diverges. Onlythe parallel beam of radiant energy reflected from the object in thescanned area is preferentially transmitted to a detector adapted toproduce electrical signals which are proportional to the reflectedenergy. The electrical signals are therefore proportional only to theradiation reflected from the thin section at the focal plane of thescanning lens system.

These signals along with signals synchronized with the x and y movementsof the lens system are transmitted to an imagedisplay device such as acathode-ray tube. Magnification is obtained because the displayed imageis made larger than the scanned area in the object. As in all opticalmicroscopes, the resolution is on the order of the wavelength of theincident radiation.

In the preferred embodiment the lens system comprises two objectivelenses, one of which is moved along the x-axis and the other of which ismoved along the y-axis to obtain the necessary scanning. The preferredsource of parallel radiant energy is a laser which may, if desired, bepulsed. in some cases the cells or tissues to be examined may bestained, although this has not been found necessary in many cases.

A preferred embodiment of the apparatus of this invention is shown inFIG. 1. The object to be examined is placed upon a support 11 which, ifdesired, may be located on a movable platform 12 having suitable meansfor making gross as well as fine adjustments in all directions. Fineadjustment of the position of the object along the optical path may beused for focusing; while gross movement of the object may be desirablefor industrial inspection purposes. Radiant energy is focused within theobject by means of the optical arrangement diagrammed in FIG. 1. A laser15 serves as a source of essentially parallel light and is powered bypower supply 16. Since lasers are available to produce essentiallyparallel radiant energy over a very broad wavelength range, the use of alaser offers a wide range of choice in radiant energy sourcecharacteristics. Moreover, the laser may be pulsed such as by pulsingthe source of radiation used to excite the laser. This may make possiblethe use of very high instantaneous light energies with the dissipatedpower being maintained below that threshold which might damage delicatetissues. Although, it is not necessary to have a source of coherentradiation, it is generally preferable since it is easier to focuscoherent radiation. It is not necessary that the radiation used bemonochromatic.

Two collimating lenses l8 and 19 are used to insure that the laser lightis completely parallel; and an adjustable iris 20 is inserted in theoptical path to eliminate any aberrations and to restrict the light beamto a desired diameter. A half-silvered mirror 21 serves as a beamsplitter and directs the light beam through a second adjustable iris 22to a pair of objective focusing lenses 25 and 26 which areantireflection coated. Lens 25 is mounted on a transducer 27 which isdriven by a suitable driving means such as an audio-oscillator 28. Thetransducer 27 and lens 25 are so arranged as to provide for thereciprocal motion of lens 25 along the y-axis of the scanning system. Inlike manner lens 26 is mounted on a transducer 30 which is driven by asuitable driving means such as an audio-oscillator 31. The transducer 30and lens 26 are so arranged as to provide for the reciprocal motion oflens 26 along the x-axis of the scanning system,

The scanning motion of the two objective lenses 25 and 26 may beachieved by a number of different means including mechanical,piezoelectric or electrostrictive devices. Although it is preferable tomove both of the lenses 25 and 26 at frequencies of the order of 50 toseveral hundred cycles per second, it is within the scope of thisinvention to scan by moving one of the lenses at a relatively slow ratesuch as by use ofa worm gear drive. The actual extent of the scanningexcursions or lens displacement will typically be between aboutone-hundredth and one millimeter.

The transducers 27 and 30 and their associated lenses 25 and 26 may inturn be mounted on a platform 32 which is movable by any suitable, knownmeans. Movable platform 32 provides another means for focusing the lightbeam within the object 10.

The combined focal point of the two vibrating lenses 25 and 26 is thepoint of scanning. A compound lens system, such as objective lenses 25and 26, behaves as a single lens. In the absence of the second lens 26,the parallel light from the laser would be focused atf,. However, withlens 26 at a distance d from lens 25, the virtual image of lens 25 actsas the object for lens 26. Thus, for lens 26, the effective focal length.r becomes (f,d) and the effective focal length s of the two lenses incombination may be shown to be represented by the expreswhere f and fare the focal lengths of lenses 25 and 26 respectively, and d is thespacing between the lenses.

1n the absence of the second lens 26. a displacement of the lens fromthe optical axis of the system by a distance 8 results in acorresponding displacement of the focal point by the same amount. in thecase of the two lens combination of FIG. 1 the displacement of eitherlens by a distance 8 produces a displacement of the focal point in thesame direction, but the displacement of the focal point is altered inmagnitude. (See FIG. 2).

The actual displacement of the focal point (8) of this compound lenssystem may be expressed as wheref andf are the focal lengths of lenses25 and 26, d is the distance between lenses and 8 is the displacement ofeither lens from the optic axis.

The placement of a N4 plate 35 between the lens system (lens 25 and 26)and the object is normally not required unless it is desired to changethe polarization of the light striking the object by from that of theincoming light. This optional M4 plate 35 is used in conjunction with anoptional analyzer 36 located in the return optical path to block out anyscattered light reflected from the optical surfaces. It has been foundthat both the M4 plate 35 and analyzer 36 are generally not requiredsince scattered light is normally not a problem in the apparatus. Inlike manner, it may be desirable under certain circumstances to usechopper 37 adjacent to the M4 plate and an interference filter 38adjacent to the analyzer, the chopper serving as a means of providing anintermittent signal to an AC detector system and the interference filterserving as an additional means to control the character of the reflectedlight transmitted to the detecting means. However, the chopper 37 andinterference filter 38 are normally not required and hence they may beconsidered as optional components in the optical system of theapparatus.

The light reflected by the object 10 under observation is directed backthrough the lens system (lenses 25 and 26) and is transmitted throughthe half-silvered mirror beam splitter 21, through a compensating glassplate 41 adapted to compensate for the displacement of the light beamproduced by the transversal of the reflected light through thehalf-silvered mirror 21 and then through a focusing,antireflection-coated objective lens 42. The lens 42 in turnpreferentially focuses the parallel component of the reflected lightthrough a pinhole 43 (which may also be an optical filter) onto aphotomultiplier tube 44 serving as a radiant energy-detecting meansadapted to produce electrical signals which are proportional to theradiant energy reflected by the object as it is scanned by the lenssystem. Since the nonparallel components of the reflected light do notreach the detector, the unwanted reflections from the object aresuppressed. Only the radiation reflected from the thin scanned sectionnear the focal plane of the scanning lens system is detected.

Power is furnished to the photomultiplier 44 from a suitable powersupply 45 and the signal output of the photomultiplier is amplified byappropriate, known equipment such as by a combination of an operationalamplifier 46 and amplifier 47. The amplified signals from the detectorare fed into the z-axis of a display means such as the cathode-ray tube48 of an oscilloscope 50. The signals generated by the reflected light,as the lenses 25 and 26 experience their scanning excursions, controlthe intensity of the display on the cathode-ray tube 48 and hence giverise to variations in the light and dark pattern 54 of the area scanned.

It is also, of course, necessary to impart motion along the xaxis andy-axis of the cathode-ray tube and to synchronize these motions withthose experienced by the lenses 25 and 26. This is accomplished byconnecting audio-oscillator 28 through an appropriate phase shifter 52to the y-axis of the tube and by connecting audio-oscillator 31 throughphase shifter 53 to the x-axis. The display 54 on the cathode-ray tube54 thereby becomes a visual image of the scanned object area.

The scanning optical microscope of FIG. 1 has been used to examineburied nerve cells at various depths. The limit of resolution using a5mW Ne-He continuous-wave laser A=6,328 A.) was about 0.5a. The depthwhich can be penetrated is governed by the wavelength of the radiationused, the intensity of the radiation and the nature of the materialbeing observed. Depths of one millimeter are relatively easy to achieveusing the Ne-He laser and it is anticipated that depths of at leastthree millimeters can be attained. Magnifications attainable are of thesame order of magnitude as those obtained by using conventional opticalmicroscopes.

FIGS. 3 and 4 illustrate another embodiment of the scanning opticalmicroscope of this invention in which a single objective lens is used tofocus the parallel light beam into the object, and in which means areprovided to move the lens rapidly in one scanning direction and slowlyin the other scanning direction. In FIGS. 3 and 4 like referencenumerals are used to refer to like components in FIG. 1.

In the embodiment of FIGS. 3 and 4 the single objective lens 60 ismounted in a support 61 which in turn is affixed to transducer 62adapted to scan along the y-axis. The transducer is in turn fastened toa platform 63 which is moved along the x-axis by any suitable drivemeans shown as-a combination of worm 64 and worm gear drive 65 driven bya motor not shown. This entire assembly of lens, transducer, platformand worm gear drive may in turn be affixed to support 66 mounted onparallel shafts 67. Support 66 may be moved by any suitable mechanism(not shown) designed to attain very fine position adjustments to focusthe beam at the desired depth in the object 10. It will, of course, beappreciated that any other means may be used to move lens 60 to achievethe desired scan excursions and that the means shown in FIGS. 3 and 4are merely exemplary of one such mechanism.

The embodiment of the apparatus of this invention shown in FIG. 5 issuitable for transmission microscopy. In FIG. 5 like components areidentified by the same reference numerals used in FIG. 1. It will beseen that the parallel radiation from laser is focused at a point withinobject 10 and then transmitted through the object by way of lenses 70and 42 to the photomultiplier 44. The focusing lens system may be formedof the combination of lenses and 26 as shown in FIG. 5 or it may beformed as a single lens as in FIG. 3. The lens system may be moved forscanning in the mode described for FIG. 1 or for FIGS. 3 and 4.

The apparatus of this invention makes possible the scanning of thinsections within thick specimens of low optical contrast. The use ofparallel light, which can be focused to a point area and reflected backas parallel light makes it possible to construct a system in which theobject to be observed is held motionless and the focusing lens system ismoved to achieve the necessary scanning. The apparatus is amenable tothe use of a relatively wide range of the electromagnetic spectrum,limited only by the ability to provide the necessary radiant energy in aparallel beam and to focus it. In the shorter wavelength, ultraviolet,it may be desirable or necessary to use quartz lenses; while in thelonger wavelength, infrared, it may be necessary to form opticalcomponents of such materials as the alkali halides, cesium iodide orother materials known to be suitable for this purpose.

The scanning optical microscope of this invention makes possible thestudy of buried cells and tissues in vivo and provides a valuableanatomical research tool.

It will thus be seen that the objects set forth above, among those madeapparent from the preceding description, are efficiently attained and,since certain changes may be made in the above constructions withoutdeparting from the scope of the invention, it is intended that allmatter contained in the above description or shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

We claim:

1. A scanning optical microscope, comprising in combination a. a sourceof parallel radiant energy;

b. optical focusing means including objective lens means adapted tofocus said radiant energy as a point source within an object to bescanned; scanning means adapted for reciprocally moving said objectivelens means of said optical focusing means along two axes orthogonal tothe optical axis;

. radiant energy-detecting means adapted to produce electrical signalsproportional to radiant energy received thereby;

optical transmission means including objective lens means adapted totransmit radiant energy from within said object to said radiantenergy-detecting means, the principal axis of said objective lens meansof said optical transmission means being parallel to the principal axisof said objective lens means of said optical focusing means; wherebyonly radiant energy reflected at the focal point of said objective lensmeans of said optical focusing means is detected; and

three-axes display means adapted to generate a visual image, the z-axisbeing connected to said radiant energydetecting means thereby to controlthe light intensity of said image, and the xand y-axes being connectedto said scanning means thereby to synchronize the x and y motions ofsaid display means with the motion of said objective lens means of saidoptical focusing means.

2. A scanning optical microscope in accordance with claim 1 wherein saidscanning means move said objective lens means of said optical focusingmeans along two mutually perpendicu lar axes.

3. A scanning optical microscope in accordance with claim 1 wherein saidsource of parallel radiant energy is a laser.

4. A scanning optical microscope in accordance with claim 1 wherein saidoptical focusing means comprises collimating lens means, beam-splittingmeans and beam diameter-restricting means in addition to said objectivelens means.

5. A scanning optical microscope in accordance with claim 4 wherein saidoptical focusing means also includes a N4 plate interposed between saidlens system and said object.

6. A scanning optical microscope in accordance with claim 1 includingmeans to move said object along the axis of the optical path of saidradiant energy, whereby the depth of scan within said object may beadjusted.

7. A scanning optical microscope in accordance with claim 1 includingmeans to move said object with respect to said scanning means.

8. A scanning optical microscope in accordance with claim 1 wherein saidobjective lens means of said optical focusing means comprises twoobjective lenses held in spaced apart relationship.

9. A scanning optical microscope in accordance with claim 8 wherein saidscanning means comprises separate transducer means associated with eachof said lenses.

10. A scanning optical microscope in accordance with claim 8 whereinsaid scanning means comprises transducer means associated with one ofsaid lenses and means to move the other of said lenses in slow mode.

1 1. A scanning optical microscope in accordance with claim 1 includingmeans to move said optical focusing means along the optical axis of saidlens system.

12. A scanning optical microscope in accordance with claim 1 whereinsaid radiant energy is reflected from said object to said radiantenergy-detecting means and said optical transmission means comprisesbeam-splitting means, beam-compensating means, and objective lens.

13. A scanning optical microscope in accordance with claim 12 in whichsaid optical transmission means also includes an optical interferencefilter and an optical analyzer.

14. A scanning optical microscope in accordance with claim 1 whereinsaid three-axis display means comprises a cathoderay tube.

15. A scanning optical microscope in accordance with claim 1 whereinsaid x-axis and y-axis of said display means are connected to saidscanning means through phase-shifter means adapted to achieve precisesynchronization between the motions of said lens means and the x and ymotions of said display means.

16. A scanning optical microscope, comprising in combination a. a laseras a source of parallel radiant energy;

b. optical means adapted to focus radiant energy as a point sourcewithin an object to be scanned and comprising in combination 1.collimating lens means,

2. beam-splitting means,

3. means to define the diameter of the beam of said radiant energy,

4. focusing lens means;

c. scanning means for imparting scanning motions to said lens meansalong two axes orthogonal to the optical axis;

d. radiant energy-detecting means adapted to produce electrical signalsproportional to radiant energy received;

e. optical means adapted to reflect radiant energy from within saidobject to said radiant energy-detecting means and comprising 1. saidbeam-splitting means,

2. beam-compensating means,

3. optical focusing means,

4. means to screen out scattered light located at said focal point ofsaid optical focusing means; and

. a cathode-ray tube-display means, the z-axis of which is connected tosaid radiant energy-detecting means and the x-axis and y-axis of whichare connected to said scanning means.

17. A scanning optical microscope in accordance with claim 16 whereinsaid focusing lens means of (b.) comprises two objective lenses held inspaced apart relationship and said scanning means comprises separatetransducer means associated with each of said lenses.

18. A scanning optical microscope in accordance with claim 16 includingmeans to move said object along the axis of the optical path of saidradiant energyQwhereby the depth of scan within said object may beadjusted.

19. A scanning optical microscope in accordance with claim 16 includingmeans to move said focusing lens means along the axis of the opticalpath of said radiant energy striking said object.

1. A scanning optical microscope, comprising in combination a. a sourceof parallel radiant energy; b. optical focusing means includingobjective lens means adapted to focus said radiant energy as a pointsource within an object to be scanned; c. scanning means adapted forreciprocally moving said objective lens means of said optical focusingmeans along two axes orthogonal to the optical axis; d. radiantenergy-detecting means adapted to produce electrical signalsproportional to radiant energy received thereby; e. optical transmissionmeans including objective lens means adapted to transmit radiant energyfrom within said object to said radiant energy-detecting means, theprincipal axis of said objective lens means of said optical transmissionmeans being parallel to the principal axis of said objective lens meansof said optical focusing means; whereby only radiant energy reflected atthe focal point of said objective lens means of said optical focusingmeans is detected; and f. three-axes display means adapted to generate avisual image, the z-axis being connected to said radiantenergy-detecting means thereby to control the light intensity of saidimage, and the x- and y-axes being connected to said scanning meansthereby to synchronize the x and y motions of said display means withthe motion of said objective lens means of said optical focusing means.2. A scanning optical microscope in accordance with claim 1 wherein saidscanning means move said objective lens means of said optical focusingmeans along two mutually perpendicular axes.
 2. beam-compensatinG means,2. beam-splitting means,
 3. means to define the diameter of the beam ofsaid radiant energy,
 3. optical focusing means,
 3. A scanning opticalmicroscope in accordance with claim 1 wherein said source of parallelradiant energy is a laser.
 4. A scanning optical microscope inaccordance with claim 1 wherein said optical focusing means comprisescollimating lens means, beam-splitting means and beamdiameter-restricting means in addition to said objective lens means. 4.means to screen out scattered light located at said focal point of saidoptical focusing means; and f. a cathode-ray tube-display means, thez-axis of which is connected to said radiant energy-detecting means andthe x-axis and y-axis of which are connected to said scanning means. 4.focusing lens means; c. scanning means for imparting scanning motions tosaid lens means along two axes orthogonal to the optical axis; d.radiant energy-detecting means adapted to produce electrical signalsproportional to radiant energy received; e. optical means adapted toreflect radiant energy from within said object to said radiantenergy-detecting means and comprising
 5. A scanning optical microscopein accordance with claim 4 wherein said optical focusing means alsoincludes a lambda /4 plate interposed between said lens system and saidobject.
 6. A scanning optical microscope in accordance with claim 1including means to move said object along the axis of the optical pathof said radiant energy, whereby the depth of scan within said object maybe adjusted.
 7. A scanning optical microscope in accordance with claim 1including means to move said object with respect to said scanning means.8. A scanning optical microscope in accordance with claim 1 wherein saidobjective lens means of said optical focusing means comprises twoobjective lenses held in spaced apart relationship.
 9. A scanningoptical microscope in accordance with claim 8 wherein said scanningmeans comprises separate transducer means associated with each of saidlenses.
 10. A scanning optical microscope in accordance with claim 8wherein said scanning means comprises transducer means associated withone of said lenses and means to move the other of said lenses in slowmode.
 11. A scanning optical microscope in accordance with claim 1including means to move said optical focusing means along the opticalaxis of said lens system.
 12. A scanning optical microscope inaccordance with claim 1 wherein said radiant energy is reflected fromsaid object to said radiant energy-detecting means and said opticaltransmission means comprises beam-splitting means, beam-compensatingmeans, and objective lens.
 13. A scanning optical microscope inaccordance with claim 12 in which said optical transmission means alsoincludes an optical interference filter and an optical analyzer.
 14. Ascanning optical microscope in accordance with claim 1 wherein saidthree-axis display means comprises a cathode-ray tube.
 15. A scanningoptical microscope in accordance with claim 1 wherein said x-axis andy-axis of said display means are connected to said scanning meansthrough phase-shifter means adapted to achieve precise synchronizationbetween the motions of said lens means and the x and y motions of saiddisplay means.
 16. A scanning optical microscope, comprising incombination a. a laser as a source of parallel radiant energy; b.optical means adapted to focus radiant energy as a point source withinan object to be scanned and comprising in combination
 17. A scanningoptical microscope in accordance with claim 16 wherein said focusinglens means of (b.) comprises two objective lenses held in spaced apartrelationship and said scanning means comprises separate transducer meansassociated with each of said lenses.
 18. A scanning optical microscopein accordance with claim 16 including means to move said object alongthe axis of the optical path of said radiant energy, whereby the depthof scan within said object may be adjusted.
 19. A scanning opticalmicroscope in accordance with claim 16 including means to move saidfocusing lens means along the axis of the optical path of said radiantenergy striking said object.